QCM-Based Measurement of Bond Rupture Forces in DNA Double

Mar 17, 2014 - INTRODUCTION. Increasing demand for cheap and rapid DNA sequencing .... thermal denaturation with the optical registration of the signa...
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QCM-Based Measurement of Bond Rupture Forces in DNA Double Helices for Complementarity Sensing Fedor N. Dultsev,*,†,§ Eugeny A. Kolosovsky,† Ivan A. Mik,†,§ Alexander A. Lomzov,‡ and Dmitrii V. Pyshnyi‡ †

Institute of Semiconductor Physics, ‡Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, 630090 Russia Novosibirsk State University, 630090 Russia

§

S Supporting Information *

ABSTRACT: After fixing the DNA molecule in the form of a double helix on the surface of a thickness shear mode resonator (QCM), mechanical oscillations at increasing amplitude cause detorsion of the helix. The force necessary for detorsion can be determined from the voltage applied to the QCM at the rupture moment. The high sensitivity of this method is due to the fact that measurements are carried out in the frequency region around the QCM resonance, where any (even very weak) distortions of the consistent oscillating system cause noticeable distortions of the amplitude-frequency dependence, and these distortions are used to fix the rupture moment. The measured rupture forces were within 30−40 pN, and the sensitivity was 108 molecules. It was demonstrated that the proposed procedure allows one to determine the factors that affect the stability of the DNA double helix. This procedure can be the basis for the development of a new method of rapid DNA analysis. Experiments performed with model DNA showed that it is possible to reveal complementarity between two DNA samples.

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INTRODUCTION Increasing demand for cheap and rapid DNA sequencing requires the development of new technologies, more rapid, cheap, and reliable. One of the examples is nanopore technology, developed for rapid and direct sequencing of separate DNA fragments that pass through a pore in the membrane and cause changes in the ion current modulation. These changes are linked with the sequence of nucleotides in DNA.1−9 However, complete sequencing is needed only rarely. In real life it is often necessary just to decide whether two polyor oligonucleotide sequences coincide or not. Is it possible to compare DNA samples for identity or complementarity without complete sequencing, just on the basis of the measured bond rupture forces? Today one of the leading methods to measure the forces of biomolecule binding is atomic force microscopy (AFM). To measure intermolecular interactions (receptor−ligands, antibody−antigens, etc.), AFM force spectroscopy experiments were applied.10−12,14,13 Intermolecular forces holding together ligand−receptor pairs are of key importance for biological processes, e.g., signaling or immune responses. Bonds underlying these processes not only play a substantial part in binding but they also provide the stability of the native molecular structure. To sustain the structure of biomolecules during their functioning, it is necessary that their intramolecular forces stabilize the three-dimensional conformation to a sufficient extent. The intramolecular stability is an obligatory condition for functioning, so there should be a substantial energy barrier © 2014 American Chemical Society

separating the native and denaturated states. How high is this barrier? Only measurements can give a correct answer. To perform such measurements, one should use a sufficiently sensitive and nonperturbing method. One such method is atomic force microscopy (AFM): functional blocks connected with DNA oligomers are bound to a complementary sequence linked to AFM tip.15−17 AFM measurements give direct data on the mechanical stability of antibody−antigen complex, on antigen resistance to isolation from the membrane, and to unfolding. It is important for the studies of intermolecular interactions that measuring method should not bring distortions into results. Interactions between molecules or their fragments comprise the key to receptor−agent systems, which is especially important in biological systems where hydrogen bonds are essential. Their energy in biopolymers ranges from 3 to 25 kcal/mol.18 To study bond rupture forces, we used a rupture event scanning method called REVS.19 The essence of this procedure is detection of particle detachment from the surface oscillating at monotonously increasing amplitude. Rupture force characteristic of a specific particle can be determined from the oscillation amplitude at which the rupture event occurs. We succeeded in determining a single virus with the help of REVS.20 Now we report measurements of the forces of interaction between Received: August 1, 2013 Revised: March 9, 2014 Published: March 17, 2014 3795

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(Sigma-Aldrich) and mercaptododecanoic acid HSCH2(CH2)9CH2COOH (thiol 2) (Sigma-Aldrich) was used for this purpose. When fixed on the surface, these compounds contain −CH3 and −COOH end groups, respectively. The ratio of thiol (1) to thiol (2) concentrations was chosen taking into account the size of DNA molecules: ∼2 nm (width) and ∼10−12 nm (length). In order to exclude the mutual effect of DNA molecules fixed on the surface, it is necessary to fix them at a distance not less than 10 nm from each other (up to 20 nm in our experiments). So, we chose the ratio of thiol (1) to thiol (2) to be 10000/1 to 40000/1. The resulting surface contains −CH3 groups and a very small concentration of −COOH groups (see Figure S1). The latter concentration must be low to ensure the degree of surface coverage by DNA molecules below 1%. This procedure is shown in more detail in Supporting Information. After the thiol monolayer was deposited, the oligomer with amino group was attached at the carboxylic group using the standard EDC/ NHS protocol. (ii) The second procedure of chemical modification is similar to that described in refs 23−26 where DNA attachment to mica surface was controlled by means of AFM. We used a similar procedure: silicon dioxide is deposited onto the QCM at the temperature of 200 °C. Then the surface is treated with K2CrO4 in sulfuric acid to achieve the higher concentration of surface OH groups. After that, the sample was placed in the atmosphere of aminopropyltriethoxysilane and incubated for 12 h at room temperature and then kept for 2 h at a temperature of 80 °C. Surface activation was performed through treatment with the solution of cyanuric chloride in anhydrous acetonitrile (4 mg/mL) for 2 h at room temperature during mixing, then the surface was washed with acetonitrile (5 times 1 mL) and dried. For oligonucleotide immobilization, a solution of biomolecules in the buffer or in 0.15 M NaCl (oligonucleotide concentration 10−5 M) was deposited on the activated surface and kept at room temperature for 12 h under humid conditions. Then the surface was washed with the buffer solution containing 0.02 M Tris/HCl pH7.5, 0.1 M NaCl, 0.002 M MgCl2, 0.05% Tween20. Thus prepared surface with immobilized biomolecules (Figure S2) was used in the studies. The second procedure gives a stronger bond of the DNA molecule with the surface. However, in the case when we used modification through thiol (see above), better controllable surface concentration was achieved; the signal generated during the rupture of the complementary oligonucleotide was narrower, and its amplitude was larger. In the case of thiol modification, the weakest bond determining DNA attachment to the surface is the gold−sulfur bond. DNA helix unwinding requires less energy, so we used mainly thiol modification because it gives a controllable DNA amount on the surface and more ordered arrangement of attached DNA molecules, excluding mutual interference since the distance between the molecules is larger than their length. Thus, a sharper signal may be achieved. The schemes explaining these two surface preparation procedures are shown in Figures S1 and S2 in Supporting Information. Oligodeoxyribonucleotides: 5′- NH2(CH2)6-p-TCAGGCAGTACCACAAGGCC-3′ (ON1) and complementary (5′GTTGCGAAAGGCCTTGTGGTACTGCCTGA-3′ (ON2) were synthesized according to the standard phosphoramidite procedure using the automatic synthesizer ACM-800. Separation was performed by means of high-performance liquid chromatography using the Agilent 1200 instrument (Agilent). The efficiency of the formation of DNA−oligonucleotide complexes was assessed also by means of thermal denaturation with the optical registration of the signal, as described in.27 In addition, we used noncomplementary sequence T20 (ON3), oligonucleotide 5′-GGCCTTATGGTACTACCTGA-3′ (ON4) having two nucleotidic discrepancies with the immobilized probe, and a fully complementary sequence 5′-GGCCTTGTGGTACTGCCTGA3′ (ON5) which forms blunt end duplex with the probe. Our study is focused on surface processes, so it is reasonable to speak of the surface concentrations. In our experiments we start with oligomer solutions 10−9−10−8 M/l and take 1 μL to deposit on the QCM surface, which makes 6 × 108−109 molecules per 1 mm2: the

biopolymer molecules (DNA) and between the fragments within one complex, which determine its structure, dynamics and functioning. Here we propose to draw attention to the possibility to use a simple procedure for DNA identification. The procedure does not require complicated analysis of DNA itself. It is based on reaching (with the help of QCM) the threshold value of the amplitude of oscillations of the QCM surface together with long molecules attached to it. At this threshold amplitude, the DNA helix starts to unfold into two complementary parts. A valuable advantage of this procedure in comparison with AFM is the absence of the need to find an object on the surface before measurement. Here we demonstrate how the REVS procedure can be used to measure bond rupture forces in a system with affine interactions, a double DNA helix as example, and to study the effect of environment composition (e.g., salt concentration) on interaction mechanism during the formation and rupture of double helices. We modified our procedure19,20 having developed a new method of rupture signal detection. Now we fix the distortions of the amplitude-frequency curve in the form of the so-called Ssignal that appears at the moment of particle detachment. The procedure has been described in.21 This allowed substantial simplification of the instrumental arrangement, and enhancement of sensitivity. Similarly to AFM, this method does not involve electromagnetic action and therefore it does not cause perturbations. The proposed procedure can serve as the basis for new types of sensors based on measuring the forces of affine interactions (probe/biomolecular target) on the surface of sensing element. To prepare the surface, we used the methods that are standard for bulk samples, but adapted them for application on surface. Now we study the interaction of DNA with modified QCM surface. Our results showed that the formation of DNA double helix can be assessed. The typical working range for the method proposed by us is 108−109 DNA molecules; this is quite comparable with the value reported by Barton for electrochemical DNA sensors:22 10−15 M, which corresponds to 6 × 108 molecules. In our experiments, we place about 108 DNA molecules on QCM surface (1 mm2), so the degree of surface coverage with DNA molecules is 1% or less. However, it is possible to use a flow system and to achieve DNA accumulation on the surface by passing the solution of lower concentration. Another important item is that our method not only detects DNA on the surface but it also measures binding forces and allows one to study the dependence of binding force on external actions, for example pH of the medium or salt content. This procedure provides the ground for the development of new types of sensors measuring the force of affine interactions in biological systems in general (e.g., probe/biomolecular target) using the surface of the sensor element. Experiments carried out with the model DNA showed that this procedure could form the basis for rapid nucleic acid testing.

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EXPERIMENTAL SECTION

We used the AT-cut quartz plates 8.25 mm in diameter, plano-convex (0.2 diopter curvature) operating at its resonance frequency of 14.3 MHz (Morion, St.Petersburg, Russia). Surface modification was carried out using two different procedures: (i) The gold electrode is deposited onto QCM, then the carboxylic group (COOH) is linked to it through the thiol group. A mixture of two thiol compounds: 1-decanethiol CH3(CH2)8CH2SH (thiol 1) 3796

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Figure 1. Scheme explaining the origin of S-signal: (a) an overview of the amplitude-frequency dependence for different values of voltage supplied to the QCM. Voltage increases stepwise from bottom to top, from 0 to 10 V. The X axis is frequency change near the resonance frequency of the QCM. Distortion corresponding to particle detachment (rupture) appears at a definite voltage value (this region is marked in plot a and shown in more detail in plot b). The S-signal is the sum of the absolute values of the integrals, or the areas between the experimental curve and the polynomial fitted to it (see the upper plot c). A set of values is obtained. The lower plot (c) is the dependence of the S-signal on the amplitude of voltage supplied to the QCM. One can see that the rupture occurs at the voltage of 2−4 V; the precise position of the maximum is determined by equating to zero the derivative of the analytical parabola y = ax2 + bx + c which is plotted point by point around the maximum. The data for ON1/ON2 unwinding (surface concentration of ON1 × 1011 molecules/mm2) are shown.

Figure 2. (a) Extreme positions of the DNA complex on the QCM surface. S-signals obtained after the first scanning of the modified surface (b): partial detachment of the linked oligomer occurs. After scanning, the complementary oligomer ON2 was added; it got bound both with the oligomer attached to the surface and with the nonbound oligomer (c). If nonbound oligomers are removed from the surface, only one signal is observed; it appears at the voltage of 3 V (d). Measurements were made on thiol-modified surface with high concentration of ON1 (5 × 1013 molecules/mm2): the solution of ON2 was added on the surface, then the surface was washed to remove nonbound ON2. degree of surface coverage with DNA molecules is thus 1%. When the surface is prepared using the first procedure, the surface concentration of DNA is determined by the thiol ratio. For the second procedure, it is determined by the concentration of the oligonucleotide. We mainly used the first procedure because thus we could control the concentration of oligonucleotides on the surface more precisely, and more uniform coverage with DNA was achieved. Below the concentrations are presented as the number of DNA molecules per unit area (mm2).

the amplitude-frequency dependence at this moment (Figure 1b). The distortion value depends on the number of detached objects, their mass and the force binding the objects to the surface. The rupture force is determined from the value of the applied voltage. The value of the distortion is called S-signal, obtained with the help of mathematical processing of the amplitude-frequency curves recorded at different voltage values (see Figure 1c). This procedure was described in more detail in ref 21. We considered the system composed of oligonucleotide 5′NH2(CH2)6-p-TCAGGCAGTACCACAAGGCC-3′ (ON1), attached to QCM surface through the amine group, and its complementary oligonucleotide 5′-GTTGCGAAAGGCCTTGTGGTACTGCCTGA-3′ (ON2). The formation of the ON1/ON2 complex was recorded. The S signal recorded in this case was broader than the signal corresponding to the rupture of uncoupled oligonucleotide ON1. In our opinion, the reason for broadening is the fact that the molecules are not arranged strictly vertically but exhibit some distribution over tilt angles. The question concerning the most probable positions of DNA molecules on the surface is still under investigation.28 In our case, the larger is deviation from the vertical position, the larger force is required to break the bonds. Figure 2 a shows

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RESULTS The procedure is based on the use of the quartz crystal microbalance as a sensor, but here the QCM acts not only as sensor but also plays an active part with respect to the particle attached to its surface. If we attach a nanometer-sized object to the QCM (AT-cut) surface (oscillating in the thickness shear mode) and supply the alternating voltage varying the frequency near the resonance (±40 kHz in our case), the QCM surface will oscillate in the shear mode with the amplitude depending on the value of applied voltage. The amplitude-frequency dependence will look as shown in Figure 1a. This dependence is described within the equivalent circuit of loaded QCM. With an increase in the amplitude of QCM surface, the particle attached to the surface gets detached, and distortions appear at 3797

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two extreme positions of DNA molecule on the surface: 1, horizontal; 2, vertical. The amplitude of surface oscillations required for rupture is lower for the vertical position than for horizontal or tilted. This is connected with the velocity gradient in liquid near the surface. Below we will consider the physical mechanism of bond rupture for the case of a long molecule fixed through its end on the surface. Figure 2b shows the result of voltage scanning for QCM with ON1 fixed on its surface, in buffer solution. A peak at about 8 V is observed. This peak corresponds to the detachment of some ON1 molecules. They remain unbound on QCM surface. Then ON2 is added, and the double helix is formed both with surface-bound ON1 and with those detached after the first scanning. Now during voltage scanning from 0 to 6 V we observe two regions: a broad peak around 3 V and a group of peaks below 1 V. This proves the presence of oligomers in two states: surface-bound and nonbound (Figure 2c). If the nonbound oligomers after the first scanning are removed by washing the surface, after the second scanning we observe only one signal at 3 V, see Figure 2d. Then the complementary oligomer ON2 was added in doses with gradual increase in the amount of the oligomer. The behavior of S-signal on the amount of ON2 is shown in Figure 3a. For the small amount of ON2 (curve 1 in Figure 3a), scanning over voltage gave a group of peaks around 0.5 V and a broad peak at 3−4 V. With an increase in ON2 amount (the resulting surface concentrations are: 2.50 × 109, 5.00 × 109, 7.50 × 109, and 1.25 × 1010 molecules/mm2, curves 1−4 in Figure 3a), the peaks change their behavior. With an increase in ON2 amount, the peak at around 0.5 V broadens, and finally a peak at 0.8 V becomes clearly pronounced. So, we see two separate peaks below 1 V. The peak at 0.8 V corresponds to the free complementary oligonucleotide. To confirm this, we performed new scanning with oligonucleotide ON3 that does not form any complex with ON1 or ON2 and has a similar molecular mass (6022 Da). In this case, we observed a signal at the voltage of 0.8 V (Figure 3a, curve 5). Here the surface washed with water and containing only ON1 molecules attached to it is termed as clean surface because this surface does not give any signal under voltage scanning to 8 V, but at the same time it is similar to the surface that was used to record curves 1−4 shown in Figure 3a.

Figure 3. (a) Peak dynamics at the voltage within the region 0.5 - 1 V, corresponding to the oligomer not bound chemically to the surface: 1−4, the amount of the added solution of complementary oligomer ON2, molecules/mm2: 2.50 × 109, 5.00 × 109, 7.50 × 109, and 1.25 × 1010, respectively; 5, nonbound complementary oligonucleotide ON3 on clean surface (1.50 × 1010 molecules/mm2). The concentration of ON1 on the surface corresponds to 1010 molecules/mm2. Thiolmodified surface. (b and c): Experimental dependencies of the helix unwinding force (the peak at 3−4 V) on experimental conditions: (b) dependence on the concentration of the oligomer attached to the surface, which confirms deviations from the vertical position. To obtain different oligomer concentrations, we varied the ratio of two thiols (thiol 1 and thiol 2) in the thiol mixture during surface preparation. (c) dependence on salt concentration. The behavior of the dependence confirms the formation of a salt shell around the double helix and thus stabilizes it. DNA surface concentration: 1.2 × 1010 molecules/mm2. Initial salt concentration: NaCl 10−6 M.

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shown in Figure 3b. As mentioned above, the peak corresponding to helix unwinding appears at the voltage of 3−4 V. Here the surface concentration was prepared using thiol modification; for the maximal DNA concentration the distance between attachment sites on the surface was 10 nm and then the distance became larger with a decrease in concentration. The force of helix unwinding depending on the concentration of salt is shown in Figure 3c. These data relate to the DNA surface concentration of 1.2 × 1010 molecules/mm2. These results confirm the formation of a salt shell around the double helix, which stabilizes it. Thermodynamic calculations carried out by the authors of refs 29−31 show that the stability of DNA duplex increases with increasing salt concentration. The authors of ref 31 demonstrated experimentally using the optical tweezers technology that the unzipping force increases with an increase in salt concentration. The data obtained by us also demonstrate an increase in the unzipping force with an increase in salt concentration. Our data are in good quantitative agreement with the reported data. Thus, the procedure proposed by us can be useful in studying the stability of DNA duplex under the effect of external factors. It should be

DISCUSSION Considering the bonding force in the double helix, we must stress that the peak that appears in the region of 3−4 V is wider, and its amplitude is higher (in comparison with a spherical object).19 The peak corresponds to unwinding of the double helix of the complementary part of oligomer bound to the surface. The width of this peak and its position are slightly dependent on oligomer concentration and on salt concentration (measurements were carried out in the buffer solution). The width of this peak may be explained by bond energy scattering in the helix and by the positions of oligomers on the surface. In the case of small concentrations, the probability of larger deviation of molecules from the vertical position is higher, which can cause changes of the force of helix unwinding. Broadening can be also connected with the mechanism of helix unwinding. However, the concentration of surface centers at which the oligomer is attached accounts for about 1%, which means that the mutual effect (interference) during rupture is insignificant. The changes of helix-unwinding force (in pN) depending on the concentration of oligomer are 3798

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Table 1. Dependence of Rupture Voltage on Oligonucleotide Used oligonucleotide

rupture voltage, V

5′-GTTGCGAAAGGCCTTGTGGTACTGCCTGA-3 (ON2) T20 (ON3) 5′-GGCCTTATGGTACTACCTGA-3 (ON4) 5′-GGCCTTGTGGTACTGCCTGA-3′ (ON5)

3 ± 0.2 0.8 ± 0.1 2 ± 0.2 3.6 ± 0.2

stressed that the instrumentation of the method proposed by us involves not so complicated sample preparation procedure as that described in ref 31. The voltage at which rupture occurs depends on binding force and on the mass of the body. We chose different oligomers to test their behavior. In our case, oligomers ON2 and ON5 differ from each other in mass, while the difference observed between ON4 and ON5 is due only to mismatches causing destabilization of the complex. Experimental data (obtained at room temperature in buffer solution at the oligomer surface concentration of 5.00 × 109 molecules/mm2) are shown in Table 1. One can see in the data presented in Table 1 that two mismatches cause essential destabilization of the complex, which we observe as rather strong difference from the case without mismatches (see Figure S3 in Supporting Information). This provides the basis for the development of a sensor to detect correspondence between two DNA or their fragments. Now the nature of DNA rupture signal and determination of bonding force are to be considered. The linear size of oligonucleotides used in the present work is 10−12 nm; molecular mass is 6232 Da (ON1) and 8965 Da (ON2), so bond rupture occurs only in liquid because inertia forces in the air are insufficient to detach the molecule from the surface. It must be stressed that the geometric size is important. With the help of QCM, high-frequency oscillations of the surface with DNA molecules fixed on it are generated. These oscillations provide the conditions for DNA unwinding. When the threshold oscillation amplitude is achieved, unwinding starts; this process is detected as the distortion of the resonance curve. In our case we have a laminar flow of the layers in liquid, Reynolds number is Re = u*L/v ≈ 1.3,32 where v is kinematic viscosity. For the QCM surface amplitude (maximal) 0.1 μm and viscosity of the liquid v = 0.01 cm2/s (this is the value for water because we work with diluted aqueous solutions), the velocity of the surface will be u0 = 850−900 cm/s, the thickness of the layer is δ = 150 nm. Figure 4 shows the plot for velocity, approximately one-third of the period, equal to 2δ. The real and imaginary parts of the velocity of liquid layers close to the surface, as well as the modulus and phase of the velocity are shown. Rather strong gradient of the velocities of different liquid layers along the DNA helix length arises. The weakest bonds in the system “DNA helix−surface” will be the first to be broken. In other words, under smoothly increasing surface oscillation amplitude, rupture starts from the weakest bond. Neglecting inertia effect, we use the solution of Navier− Stokes equation for a small particle with diameter d (Reynolds number: Re = ρd(ωu0)/η ≈ 1) in a continuous viscous liquid. Then the force applied to the center of gravity of the particle is equal to

Figure 4. Dependence of the velocity of the liquid above the QCM surface on the distance from the surface; the velocity gradient explains the nature of the rupture force. The solution is given for a molecule shaped as ellipsoid.

large molecule, or the direction of liquid movement and the geometry of the body: the molecule is considered to be an oblong ellipsoid. Then the friction coefficient is:32,33 ξ=

6π rη0L p2 − 1 p ln

p + p2 − 1 p − p2 − 1

where p = L/d is the asymmetry parameter of the particle shape (L is the length of the particle and d is its diameter). We take p = 6, see Figure 4b. ξ = 6πηL 0.1989

or Ra2 =

1 [3bc + a(b + c)] 5

It is assumed here and below that the axle is parallel to the direction of the incident flow. Then, the force affecting the gravity center of the ellipsoid is ⎯ → Fc =

⎯ → Fc =

F ⃗ = 6π rηV⃗ Here η is viscosity coefficient (dynamic viscosity). In this solution, important part is played by the hydrodynamics of a

∫x

L

ξV⃗ dx

0

ξu0 L − x0

∫x

L

exp(i(1 + i)x /δ)/dx 0

Rupture force values are shown in Table 2. They rather well correlate with the reported AFM data.15 Typical rupture force 3799

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Table 2. Calculated Values of Rupture Forces for Experimentally Determined Voltage Values (Amplitude of Surface Oscillations) U, V

molecular mass, a.m.u.

L, nm, molecule length

V, m/s

F, pN, corrected for shape

0.5 0.8 3

6232 + 8965 6022 8965

12 12 12

0.58 0.93 3.49

13.1 ± 2.0 11 ± 2.0 36.0 ± 3.0

Figure 5. Scheme of the test for the correspondence between two DNA samples.

values are several tens pN for complexes of 20 pairs,16 and this coincidence makes us suppose the similarity of rupture mechanisms. It seems probable that the rupture starts from the outer end, similarly to zipper opening. This result confirms the fact that not all DNA molecules on the surface are strictly vertical; tilt means different effective cross sections of the molecules, which leads to peak broadening. This is especially clear at low DNA concentrations, while at high concentrations the fraction of strictly vertical molecules becomes larger. Using the model of two states,34 we considered the formation of a bimolecular complex of oligonucleotides and determined the thermodynamic parameters of the formation of double helix structure. Calculated changes of the Gibbs energy for this system of oligomers increase with an increase in salt concentration. Comparing the experimental data with calculation results, we estimate that 1 pN corresponds to 0.64 kcal/ mol (without salt addition; at room temperature). The method proposed by us may be the ground for rapid DNA analysis. It may be used to detect the complementarity of DNA sequences. According to our measurements, the sensitivity of this method is not worse than 108 molecules. On the basis of results obtained, two procedures can be proposed to reveal the target sequences in DNA. The first procedure relies on the principle of the proposed sensor, which is to distinguish between single and double DNA helices adsorbed on the surface, on the basis of different masses of these components. Either complementary oligomer for the DNA under determination or the DNA itself are placed on the QCM surface. It is desirable that the molecular masses of these complementary oligomers are approximately equal. This will allow us to obtain more distinct signals either in the absence of DNA to be determined, or during DNA determination. Otherwise peak broadening will be observed. Complementary pairs placed on the surface should be weakly bound to the surface; only one narrow peak corresponding to the detachment of DNA molecule will be observed during scanning. As an example, we will consider the same system as that described above. DNA molecules are placed on the QCM surface coated with silicon dioxide. A signal at the voltage of about 1 V is observed during scanning. If DNA placed on the surface gets bound with its complementary pair (a double helix will be formed), we will see a different picture. The second peak will appear at lower voltage (about 0.5 V). The second procedure can be used to test two DNA species for identity or complementarity. The experimental procedure is illustrated schematically in Figure 5. For this purpose, we fix DNA-1 on the QCM surface, scan voltage up to 5−6 V, then wash the surface to remove the detached complementary oligonucleotide. In other words, we make a single helix from the double helix. Thus we make a detection cell. The DNA-x, which is to be tested for identity, is fixed on the surface of another QCM. Then we carry out the treatment, perform detachment of the complementary oligonucleotide. After that,

we transfer this solution with the complementary oligonucleotide to the detection cell with DNA-1. Now two versions of scanning results can appear: in the case of complete identity between DNA-1 and DNA-x, we will observe the signal at the voltage of 3−4 V (helix unwinding), while in the case of the complete absence of any correspondence we will observe a signal at about 1 V (unbound complementary oligonucleotide from DNA-x). This was tested experimentally with model DNA samples. In the case of unconformity we observed the signal at the voltage of 1 V, while in the case of identity the signal appeared at 3 V. The detection cell can be used many times. Thus, the first steps have been made toward the development of a prototype of electromechanical sensor of nucleic acid hybridization. Investigation aimed at preparing the sensor surface was carried out, and the protocols for immobilization of nucleotide probes were developed. The same functionalized surface can serve as a sensor for different DNA, while identification can be done on the basis of the value of rupture force for a complementary pair.

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CONCLUSIONS The sensitivity of the method allows one to detect DNA molecules with the surface concentration about 108 molecules per 1 mm2. Detachment of DNA molecule from the QCM surface occurs not due to inertia forces as was the case for large particles (>150 nm) but due to the velocity gradient in the liquid, which is formed as a result of the harmonic oscillations of the QCM surface. Because the measurements of distortions in the amplitude-frequency dependence are performed near the resonance region of QCM oscillations, any slightest disturbances of the consistent oscillation system (e.g., detachment of macromolecules from the surface) cause noticeable distortions of the amplitude-frequency dependence. This defines the high sensitivity and simplicity of the procedure. We carried out direct measurement of the bond rupture force in the DNA double helix. The data confirmed that the bonding force in the double helix of DNA under investigation was equal to 30−40 pN. The bond rupture force depends on salt concentration; the stability of the double helix increases with an increase in salt concentration. Experiments performed with model DNA samples showed that this procedure may serve as the basis for the development of the tools for rapid DNA analysis. It may also be useful in studying other kinds of interactions in biological systems including, for example, antibody recognition or protein-drug interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Surface preparation procedures and S-signals for different kinds of oligonucleotides shown in Table 3 are presented in 3800

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Supporting Information. This information is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address

F.N.D.: Institute of Semiconductor Physics, SB RAS, Lavrentyev ave., 13, 630090, Novosibirsk, Russia. Author Contributions

F.N.D. authored the idea of the method, designed experiments, made surface modifications, performed data analysis, coauthored the manuscript, and is responsible for the overall quality of the work. E.A.K. made the software for processing experimental results. I.A.M. conducted measurements and made preliminary treatment of the results. A.A.L. and D.V.P. synthesized oligonucleotides, made surface modifications, and coauthored the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Integration Project of SB RAS (No. 86), and by the Ministry of Education and Science of the Russian Federation.

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ABBREVIATIONS REVS rupture event virus scanning; QCM quartz crystal microbalance; AFM atomic force microscopy; ON1 5′- NH2(CH 2 ) 6 -p-TCAGGCAGTACCACAAGGCC-3′; ON2 5′GTTGCGAAAGGCCTTGTGGTACTGCCTGA-3′; ON3 T20; ON4 5′-GGCCTTATGGTACTACCTGA-3′; ON5 5′GGCCTTGTGGTACTGCCTGA-3′

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REFERENCES

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dx.doi.org/10.1021/la402971a | Langmuir 2014, 30, 3795−3801